Much interest has been directed at understanding the adjuvant properties of the heat-labile enterotoxin of Escherichia coli (LT). In this study, we have assessed how LT compared with the nonenzymatic mutant LT (E112K) affect the level of B7-1 and B7-2 expression on APCs, and we determined how these costimulatory molecules influence their adjuvant properties. Analysis of B7-1 and B7-2 expression on B cells revealed that LT enhanced B7-2 but not B7-1, while LT (E112K) had no effect on the expression of either costimulatory molecule. Treatment of macrophage or dendritic cells with LT resulted in a predominant enhancement of B7-2, while LT (E112K) induced mainly B7-1 expression. Analysis of LT- and LT (E112K)-treated B cells, macrophage, and dendritic cells also revealed significant differences in their ability to enhance anti-CD3-stimulated CD4+ T cell proliferative responses via B7-1 and B7-2. Furthermore, the ability of LT to enhance both Ab and CD4+ T cell responses to a coadministered Ag was severely abrogated in B7-2- but not B7-1-deficient mice. In contrast, the in vivo adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4+ T cell responses, while B7-1 appeared to be the predominant B7 molecule involved in the ability of LT (E112K) to augment Ab responses to a coadministered Ag. These findings demonstrate distinct differences in the ability of LT and LT (E112K) to enhance B7-1 and B7-2 on APC, as well as a dependence upon these costimulatory molecules for their adjuvant properties.

The heat-labile enterotoxin of Escherichia coli (LT)3 is an oligomeric toxin consisting of an enzymatically active A subunit that ADP-ribosylates the Gsα subunit, thereby irreversibly activating adenylate cyclase, and a pentameric B subunit that is responsible for the binding properties of LT to cell surface gangliosides (1, 2, 3, 4). Previous studies assessing the adjuvant properties of LT and the closely related cholera toxin (CT) have shown that these molecules act as potent mucosal and systemic adjuvants (5, 6, 7). However, due to their inherent toxicity, various strategies have been used to reduce or abrogate their enzymatic properties while concurrently maintaining their adjuvant properties.

Mutants of LT have been generated that contain a single amino acid substitution within the A subunit and possess nondetectable levels of ADP-ribosyltransferase activity (8, 9, 10, 11). These molecularly defined, nonenzymatic mutants have facilitated studies to explore mechanisms by which LT acts as a mucosal and systemic adjuvant. Studies directly comparing LT and nonenzymatic mutants of LT have shown that these molecules are capable of priming CD4+ T cells that provide B cell help for the induction of Ag-specific mucosal IgA and serum IgG Ab responses (8, 9, 10, 11, 12). However, assessment of the CD4+ Th cell profiles has suggested that there are significant differences in the ability of the native and nonenzymatic mutants of LT to prime Th cell subsets (8, 11). In this regard, native LT has been shown to predominantly up-regulate the costimulatory molecule, B7-2, whereas a mutant LT enhanced B7-1 expression on peritoneal macrophage (11). Moreover, the ability of LT to enhance B7-2 expression on peritoneal macrophage was associated with ADP-ribosyltransferase activity. Due to the importance of the B7-CD28 signaling pathway for optimal CD4+ T cell activation and effector function (13, 14, 15, 16, 17), these studies suggest that native and nonenzymatic mutants of LT may be differentially using B7 molecules for their adjuvant properties. However, it is presently unclear whether differences exist in the ability of native and mutant LT to enhance B7-1 and B7-2 expression on other APC, including B cells and dendritic cells, and what functional role these costimulatory molecules play in the ability of LT and nonenzymatic mutants of LT to act as adjuvants.

The aim of the present study was to assess the effect of native LT and of the nonenzymatic mutant LT (E112K) on B7-1 and B7-2 expression on APC and how these costimulatory molecules affected their mucosal and systemic adjuvant properties. The results obtained from this study demonstrate that LT and LT (E112K) differentially induced B7-1 and B7-2 expression on B cells, macrophage, and dendritic cells and resulted in unique differences in the ability of LT- and LT (E112K)-treated APC to enhance CD4+ T cell proliferative responses in vitro. Our in vivo studies using B7-1- or B7-2-deficient mice provide evidence that the adjuvant properties of native LT involved up-regulation of B7-2 but not B7-1 expression. Furthermore, the in vivo adjuvant properties of LT (E112K) required both B7-1 and B7-2 expression for optimal CD4+ T cell responses, whereas B7-1 was predominantly responsible for enhanced systemic and mucosal Ab responses to a coadministered Ag.

The recombinant SBR from Streptococcus mutans was purified as previously described (18). LT and LT (E112K) were produced and purified as described by Cheng et al. (8). Analysis of LT and LT (E112K) for endotoxin content was performed by means of a Quantitative Chromagenic Limulus Amebocyte Lysate assay kit (BioWhittaker, Walkersville, MD) using E. coli K235 LPS as standard. The endotoxin content was <40 pg/mg LT or LT (E112K).

Mice, 8–12 wk old, were maintained in a pathogen-free facility at the University of Alabama (UAB; Birmingham, AL). The generation of B7-1- and B7-2-deficient BALB/c mice was previously described (19). Wild-type, B7-1−/−, and B7-2−/− BALB/c mice were maintained and bred within the animal facility at UAB. Groups of six to eight animals were immunized by the intranasal (i.n.) route on days 0 and 18 with PBS, 20 μg of SBR alone, or 20 μg of SBR supplemented with 5 μg of LT or LT (E112K). All studies were approved by the UAB Institutional Animal Care and Use Committee.

Samples of saliva and plasma were collected from individual mice 1 day before the first immunization (day 0) and 2 wk following the last i.n. immunization. Saliva was collected after stimulation of salivary flow by injecting each mouse i.p. with 5 μg of carbachol (Sigma-Aldrich, St. Louis, MO) in 0.1 ml of PBS. Plasma samples were obtained following centrifugation of blood collected from the retroorbital plexus using a calibrated heparinized capillary tube. Mucosal secretions and plasma samples were stored at −70°C and −20°C, respectively, until assayed for Ab activity.

Levels of isotype-specific Abs in saliva and plasma were quantitated by ELISA. Polystyrene microtiter 96-well plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 2.5 μg/ml SBR. Total IgA levels in the saliva were determined by coating plates with goat anti-mouse IgA Abs (Southern Biotechnology Associates, Birmingham, AL). Serial 2-fold dilutions of plasma or saliva were added in duplicate and plates were incubated overnight at 4°C. Plates were then washed with PBS containing 0.1% Tween (PBS-Tw) and incubated at room temperature with the appropriate peroxidase-conjugated goat anti-mouse Ig isotype-specific reagent (Southern Biotechnology Associates). After washing, plates were developed with o-phenylenediamine and hydrogen peroxide for 20 min and OD490 was measured. The levels of Abs and of total Ig were calculated by interpolation on calibration curves generated by using a mouse Ig reference serum (ICN Biomedicals, Aurora, OH).

Splenic CD4+ T cells were negatively selected by using the Mouse CD4+ Subset Column according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN). The purity of the recovered CD4+ T cells was >90%. B220+ cells were obtained from cervical lymph nodes (CLN) or spleen by using CD43 MicroBeads (Miltenyi Biotec, Sunnyvale, CA). CD43 MicroBeads were incubated with a cell suspension for 15 min at 6°C in PBS containing 0.5% BSA and 2 mM EDTA. The cell suspension was then washed by adding a 20-fold excess of PBS containing 0.5% BSA and 2 mM EDTA. After centrifugation, the cells were resuspended in 1 ml PBS-0.5% BSA and added to the magnetic depletion column. B cells were eluted with 15 ml of PBS containing 0.5% BSA and 2 mM EDTA. This procedure routinely yielded >99% B220+ cells as shown by flow cytometry. CD11b+ cells were isolated from CLN and spleen by using the CD11b Microbead System according to the manufacturer’s protocol (Miltenyi Biotec). The positively selected cells were rested overnight in polypropylene tubes before being used in experiments. The purity of CD11b-selected cells were typically >85%, as determined by flow cytometry. For use in the CD4+ T cell coculture assay, dendritic cells were generated from the bone marrow of BALB/c mice by culturing bone marrow cells in the presence of 20 ng/ml rGM-CSF for 10 days in complete medium (RPMI 1640 containing 10% FBS, 2 mM l-glutamine, 50 μM 2-ME, 20 mM HEPES, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 μg/ml streptomycin) (20). The FBS contained <0.06 EU/ml (Cellgro Mediatech, Washington, DC). Analysis of the resulting cell population demonstrated that >70% of the cells stained positive for CD11c expression.

To assess the ability of LT or LT (E112K) to up-regulate the costimulatory molecules B7-1 and B7-2 on B220+, CD11b+, or CD11c+ cells from a mucosal IgA inductive site, a single-cell suspension obtained from the nasal-associated lymphoid tissue (NALT) (21) was incubated with 1 μg/ml LT or LT (E112K) in complete medium. Briefly, the lower jaws of euthanized mice were removed and the NALT-containing tissue was isolated from the nasal cavity using a scalpel and cutting along the inside edge of the hard palate. The tissue was removed from the roof of the mouth and the nonencapsulated bilateral strip of lymphoid tissue (NALT) was scraped away and passed through a syringe to create a single-cell suspension. The resulting cell suspension was then subjected to a Histopaque (SG-1.083) density gradient to remove tissue debris and RBCs. Following a 24-h incubation, cells were incubated for 15 min in PBS containing 0.1% sodium azide and 3% FBS (FACS buffer), washed, and stained with allophycocyanin-labeled B220, CD11b, or CD11c mAb and then with FITC-labeled B7-1 and PE-labeled B7-2 mAbs (BD PharMingen, San Diego, CA). Cells were then washed twice with FACS buffer, fixed with 2% paraformaldehyde, and analyzed by FACScan (BD Biosciences, Sunnyvale, CA).

To assess differences in the functional costimulatory activity of B7-1 and B7-2 on LT- or LT (E112K)-treated APC, purified B220+, CD11b+, and CD11c+ cells were treated with medium alone, LT, or LT (E112K) for 24 h, washed in ice-cold PBS, fixed in 0.5% paraformaldehyde, and washed three times with PBS containing 10% FBS. Cultures of CD4+ T cells (2 × 106 cells/ml) in complete medium were cultured with 0.5% paraformaldehyde-fixed B220+, CD11b+, or CD11c+ cells (4 × 106 cells/ml) and 10 ng/ml anti-CD3 mAb in the presence or absence of functional grade mAbs to B7-1, B7-2, or isotype control Abs for 5 days (eBioscience, San Diego, CA). Approximately 20 h before harvesting, the cell cultures were pulsed with 0.5 μCi of [3H]thymidine and cpm were determined by using a liquid scintillation counter.

Purified CD4+ T cells (2.5 × 106/ml) from the CLN or spleen were cocultured with gamma-irradiated (2500 rad) splenic feeder cells (4 × 106/ml) in complete medium. Medium alone or SBR (2.5 μg/ml) was added to cells and cultures were incubated for 4 days in a humidified CO2 incubator at 37°C. The levels of IL-4, IL-5, and IFN-γ in culture supernatants were determined using ELISA kits obtained from BD PharMingen. CD4+ T cell proliferation was measured by adding 0.5 μC of [3H]thymidine for the last 20 h of culture. The incorporation of [3H]thymidine was determined using a liquid scintillation counter; data are expressed as cpm of experimental (SBR-stimulated) minus the medium-treated controls.

Statistical significance between groups was evaluated by ANOVA and the Tukey multiple-comparison test using the InStat program (GraphPad, San Diego, CA). Differences between groups were considered significant at the level of p < 0.05.

To assess the effects of LT and LT (E112K) on B7-1 and B7-2 expression by APC isolated from the mucosal inductive site NALT, a single-cell suspension of cells was treated with a concentration range (1–10,000 ng/ml) of either LT or LT (E112K) for 24 h and then analyzed for B7 expression (Fig. 1). Analysis of B7-1 and B7-2 expression on B (B220+) cells revealed that LT selectively enhanced B7-2 expression (p < 0.05; Fig. 1, A and B). At all concentrations tested, LT (E112K) did not enhance the levels of B7-1 or B7-2 on B cells as compared with nonstimulated control cells (Fig. 1, A and B). Assessment of B7-1 and B7-2 expression on macrophage revealed that LT and LT (E112K) induced similar levels of B7-1 expression on macrophages (Fig. 1,C). The level of B7-2 expression on LT-treated macrophages was increased >4-fold, whereas LT (E112K) increased B7-2 expression by ∼2-fold, but only at concentrations >100 ng/ml (Fig. 1,D). The ability of LT and LT (E112K) to up-regulate B7-1 and B7-2 on dendritic cells revealed a different pattern (Fig. 1, E and F). B7-1 expression on dendritic cells was minimally affected by LT treatment while LT induced a >5-fold increase in the level of B7-2 expression (Fig. 1, E and F). In contrast, LT (E112K) enhanced predominantly B7-1 expression on dendritic cells (Fig. 1, E and F). A similar pattern was observed with cells incubated for 48 or 72 h (data not shown). These results demonstrate that LT and LT (E112K) differentially enhance B7-1 and B7-2 expression on different types of APC.

FIGURE 1.

Expression of B7-1 and B7-2 on B220+ cells (A and B), CD11b+ cells (C and D), and CD11c+ cells (E and F) from NALT. A single-cell suspension isolated from NALT was incubated in the presence or absence of various concentrations of LT or LT (E112K) for 24 h and then analyzed for B7-1 (A, C, and E) and B7-2 (B, D, and F) expression, respectively, by flow cytometry. Similar results were obtained at 48- and 72-h time points (data not shown). ∗, Significant differences (p < 0.05) compared with nonstimulated (medium) control. The data are expressed as the mean of five separate experiments. SD bars were omitted for clarity.

FIGURE 1.

Expression of B7-1 and B7-2 on B220+ cells (A and B), CD11b+ cells (C and D), and CD11c+ cells (E and F) from NALT. A single-cell suspension isolated from NALT was incubated in the presence or absence of various concentrations of LT or LT (E112K) for 24 h and then analyzed for B7-1 (A, C, and E) and B7-2 (B, D, and F) expression, respectively, by flow cytometry. Similar results were obtained at 48- and 72-h time points (data not shown). ∗, Significant differences (p < 0.05) compared with nonstimulated (medium) control. The data are expressed as the mean of five separate experiments. SD bars were omitted for clarity.

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To determine whether the level of B7 expression induced by LT or LT (E112K) on APC had an effect on inducing a functional costimulatory signal, LT- or LT (E112K)-treated APC were coincubated with anti-CD3-stimulated CD4+ T cells in the presence or absence of mAbs to B7-1 or B7-2 (Fig. 2). Cultures containing LT-treated B cells showed significantly (p < 0.05) enhanced B7-2 (nonstimulated mean fluorescence intensity (MFI) = 198 ± 78; LT-treated MFI = 1141 ± 347) but not B7-1 (nonstimulated MFI = 113 ± 42; LT-treated MFI = 133 ± 79) expression, and significantly (p < 0.05) higher CD4+ T cell responses compared with the medium-treated control cultures (Fig. 2,A). The addition of anti-B7-2 but not anti-B7-1 mAb to LT-treated B cells resulted in a significant (p < 0.05) decrease in the proliferative response of CD4+ T cells (Fig. 2,A). LT (E112K)-treated B cells induced a modest but insignificant (p > 0.05) increase in T cell proliferation (Fig. 2,A). Moreover, the addition of anti-B7-1 or B7-2 mAb to these cultures resulted in no significant inhibitory effect on CD4+ T cell proliferation (Fig. 2 A). These findings are in agreement with the inability of LT (E112K) to enhance either B7-1 (nonstimulated MFI = 113 ± 42; LT (E112K)-treated MFI = 119 ± 66) or B7-2 (nonstimulated MFI = 198 ± 78; LT (E112K)-treated MFI = 188 ± 93) expression.

FIGURE 2.

LT- or LT (E112K)-treated APCs induce enhanced CD4+ T cell proliferation. B220+ (A), CD11b+ (B), and CD11c+ (C) were pretreated with medium alone or 1 μg/ml LT or LT (E112K) for 24 h. After pretreatment, cells were then fixed with 0.5% paraformaldehyde, washed, and added to purified CD4+ T cells in the presence of anti-CD3 (10 ng/ml) stimulation and the presence or absence of inhibitory Abs to B7-1 or B7-2. Cultures were incubated for 120 h and pulsed with [3H]TdR during the last 20 h of the incubation. ∗, Significant differences (p < 0.05) compared with no inhibitory Ab. The results were expressed as the mean ± SD of three separate experiments.

FIGURE 2.

LT- or LT (E112K)-treated APCs induce enhanced CD4+ T cell proliferation. B220+ (A), CD11b+ (B), and CD11c+ (C) were pretreated with medium alone or 1 μg/ml LT or LT (E112K) for 24 h. After pretreatment, cells were then fixed with 0.5% paraformaldehyde, washed, and added to purified CD4+ T cells in the presence of anti-CD3 (10 ng/ml) stimulation and the presence or absence of inhibitory Abs to B7-1 or B7-2. Cultures were incubated for 120 h and pulsed with [3H]TdR during the last 20 h of the incubation. ∗, Significant differences (p < 0.05) compared with no inhibitory Ab. The results were expressed as the mean ± SD of three separate experiments.

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Macrophages treated with LT showed enhanced expression of both B7-1 (nonstimulated MFI = 291 ± 106; LT-treated MFI = 941 ± 256) and B7-2 (nonstimulated MFI = 613 ± 249; LT-treated MFI = 2891 ± 488) expression and supported enhanced CD4+ T cell proliferation that was predominantly mediated by B7-2. LT (E112K)-treated macrophages showed increased levels of both B7-1 (nonstimulated MFI = 291 ± 106; LT (E112K)-treated MFI = 1177 ± 329) and B7-2 (nonstimulated MFI = 613 ± 249; LT (E112K)-treated MFI = 1460 ± 218) expression and supported a significantly (p < 0.05) enhanced T cell proliferative response that was dependent upon both B7-1 and B7-2 (Fig. 2,B). Assessment of LT- and LT (E112K)-treated dendritic cells also revealed differences in the costimulatory activity provided by B7-1 and B7-2. LT-treated bone marrow-derived dendritic cells exhibited no significant increase in B7-1 expression (nonstimulated MFI = 498 ± 138; LT-treated MFI = 521 ± 177). In contrast, dendritic cells coincubated with LT exhibited a >4-fold increase in B7-2 expression (nonstimulated MFI = 898 ± 338; LT-treated MFI = 3782 ± 584) and a marked increase in CD4+ T cell proliferation that was significantly (p < 0.05) inhibited by anti-B7-2 but not anti-B7-1 mAb (Fig. 2 C). In contrast, the enhanced proliferative responses observed with LT (E112K)-treated dendritic cells appeared to be mediated by both B7-1 and B7-2. This was in agreement with the ability of LT (E112K) to enhance both B7-1 (nonstimulated MFI = 498 ± 138; LT (E112K)-treated MFI = 2477 ± 633) and B7-2 (nonstimulated MFI = 898 ± 338; LT (E112K)-treated MFI = 1811 ± 397) expression on dendritic cells. These results indicate that LT- and LT (E112K)-treated APC support different costimulatory activity via B7-1 and B7-2 to anti-CD3-stimulated CD4+ T cells.

To determine the effect of LT- or LT (E112K)-induced B7-1 and B7-2 expression on their adjuvant properties, wild-type BALB/c mice or BALB/c mice deficient in B7-1 or B7-2 were immunized i.n. with SBR alone or with LT or LT (E112K) as adjuvant. B7-2-deficient mice immunized with SBR and LT exhibited a >70% reduction in both plasma IgG and IgA anti-SBR responses as compared with wild-type control mice receiving SBR and LT (Fig. 3, A and C). In contrast, no differences were seen in the levels of plasma IgG or IgA anti-SBR from B7-1-deficient mice immunized with SBR and LT as adjuvant compared with wild-type control mice (Fig. 3, A and C).

FIGURE 3.

Augmentation of anti-SBR Ab responses by LT or LT (E112K) in wild-type (wt), B7-1-, or B7-2-deficient BALB/c mice immunized by the i.n. route. Plasma IgG (A and B), IgA (C and D), or salivary IgA (E and F) anti-SBR responses were assessed 3 wk after the last immunization. Ab responses to SBR and total Ig levels were determined by ELISA. Data are expressed as the arithmetic mean ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

FIGURE 3.

Augmentation of anti-SBR Ab responses by LT or LT (E112K) in wild-type (wt), B7-1-, or B7-2-deficient BALB/c mice immunized by the i.n. route. Plasma IgG (A and B), IgA (C and D), or salivary IgA (E and F) anti-SBR responses were assessed 3 wk after the last immunization. Ab responses to SBR and total Ig levels were determined by ELISA. Data are expressed as the arithmetic mean ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

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Assessment of the ability of LT (E112K) to enhance plasma IgG and IgA responses to SBR revealed a >50% reduction in B7-1- but not B7-2-deficient mice (Fig. 3, B and D). To further determine the role of B7-1 and B7-2 in the mucosal adjuvant properties of LT and LT (E112K), the levels of saliva IgA anti-SBR were measured. A similar profile was observed as that seen with the plasma IgA and IgG responses to SBR. The ability of LT to augment saliva IgA responses to SBR were severely reduced in B7-2-deficient mice; however, LT was capable of enhancing saliva IgA anti-SBR responses in B7-1-deficient mice to a level similar to that observed in wild-type mice immunized with SBR and LT (Fig. 3,E). In contrast, the ability of LT (E112K) to enhance saliva IgA anti-SBR responses was predominantly attributed to B7-1 (Fig. 3,F). In this regard, no significant increase in the level of saliva IgA anti-SBR was seen in B7-1-deficient mice receiving SBR and LT (E112K) as compared with wild-type mice receiving SBR alone (Fig. 3 F). Taken together, these data demonstrate that LT and LT (E112K) differentially up-regulate B7-1 and B7-2 expression for enhancing both systemic and mucosal Ab responses to a coadministered Ag.

A previous study using CT as adjuvant demonstrated that blocking B7-2 in vivo resulted in selective decrease in IgG1 but not IgG2a Ab levels to a coadministered Ag (22). Thus, we next wanted to determine whether there was a similar decrease in plasma anti-SBR IgG1 or IgG2a responses in B7-1- or B7-2-deficient mice, respectively, that accounted for the observed decreased plasma IgG Ab response to SBR. Wild-type mice receiving LT or LT (E112K) as adjuvant had significantly increased levels of plasma anti-SBR IgG1, IgG2a, and IgG2b as compared with mice immunized with SBR alone (Fig. 4). Moreover, B7-2-deficient mice immunized with LT as adjuvant exhibited significantly decreased (p < 0.05) levels of anti-SBR IgG1, IgG2a, and IgG2b (Fig. 4, A, C, and E). However, no significant decrease in anti-SBR IgG1, IgG2a, or IgG2b was observed in B7-1-deficient mice immunized with SBR and LT (Fig. 4, A, C, and E). Analysis of the IgG subclass response from mice immunized with LT (E112K) as adjuvant demonstrated that the ability of LT (E112K) to augment anti-SBR IgG1, IgG2a, and IgG2b responses was predominantly dependent upon B7-1 but not B7-2 expression (Fig. 4, B, C, and F). Thus, while no selective reduction was observed in the IgG1 or IgG2a response to SBR in B7-2- or B7-1-deficient mice, respectively, these results suggest that LT up-regulates B7-2 to enhance IgG subclass responses to SBR, while LT (E112K) used B7-1.

FIGURE 4.

Plasma IgG1 (A and B), IgG2a (C and D), and IgG2b (E and F) subclass Ab responses to SBR from mice immunized by the i.n. route with SBR alone or with LT or LT (E112K) as adjuvant. Plasma samples were collected 3 wk after the last immunization. Data represent the arithmetic means ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

FIGURE 4.

Plasma IgG1 (A and B), IgG2a (C and D), and IgG2b (E and F) subclass Ab responses to SBR from mice immunized by the i.n. route with SBR alone or with LT or LT (E112K) as adjuvant. Plasma samples were collected 3 wk after the last immunization. Data represent the arithmetic means ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

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While the effect of B7-1 and B7-2 molecules affecting CD4+ Th cell profiles remains controversial, optimal T cell activation and effector functions are initially dependent upon B7 costimulation (13, 14, 15, 16, 17, 22). Therefore, to assess how the ability of LT or LT (E112K) to enhance B7 molecules on APC was affecting the proliferation and cytokine production of SBR-specific CD4+ T cells, purified splenic CD4+ T cells from mice i.n. immunized with SBR alone or with LT or LT (E112K) as adjuvant were restimulated in vitro with SBR and wild-type APC. SBR-specific CD4+ T cell proliferation from wild-type mice immunized with LT or LT (E112K) as adjuvant exhibited a significant enhancement in proliferation compared with wild-type mice receiving SBR alone (Fig. 5, A and B). Moreover, a >45% decrease was observed in CD4+ T cell proliferation in B7-2-deficient mice immunized with SBR and LT as compared with wild-type controls (Fig. 5,A). However, no significant decrease in T cell proliferative responses was observed in B7-1-deficient mice immunized with LT as adjuvant. In contrast, both B7-1- and B7-2-deficient mice receiving LT (E112K) as adjuvant exhibited decreased CD4+ T cell responses to SBR compared with wild-type mice immunized with SBR and LT (E112K) (Fig. 5 B).

FIGURE 5.

Proliferation responses (A and B) and Th1- and Th2-type cytokine production (CE) by SBR-specific CD4+ T cells isolated 4 wk after the last immunization of mice immunized by the i.n. route with SBR alone or with LT or LT (E112K) as adjuvant. CD4+ T cells (2 × 106/ml) were isolated from spleen and were cocultured with irradiated, T cell-depleted splenic feeder cells (4 × 106/ml) in the presence or absence of various concentrations of SBR. [3H]TdR was added during the last 20 h of a 96-h culture. The amount of [3H]TdR incorporated was measured using a liquid scintillation counter. Culture supernatants were harvested after 96 h and analyzed for cytokines by ELISA. Results are expressed as the arithmetic mean ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

FIGURE 5.

Proliferation responses (A and B) and Th1- and Th2-type cytokine production (CE) by SBR-specific CD4+ T cells isolated 4 wk after the last immunization of mice immunized by the i.n. route with SBR alone or with LT or LT (E112K) as adjuvant. CD4+ T cells (2 × 106/ml) were isolated from spleen and were cocultured with irradiated, T cell-depleted splenic feeder cells (4 × 106/ml) in the presence or absence of various concentrations of SBR. [3H]TdR was added during the last 20 h of a 96-h culture. The amount of [3H]TdR incorporated was measured using a liquid scintillation counter. Culture supernatants were harvested after 96 h and analyzed for cytokines by ELISA. Results are expressed as the arithmetic mean ± SD of results from six mice per group. ∗, Significant differences (p < 0.05) compared with wild-type control animals receiving SBR with adjuvant.

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SBR-specific CD4+ T cells also revealed distinct differences in Th1- and Th2-type cytokine production from mice immunized with LT or LT (E112K) as adjuvant. Wild-type mice immunized with LT or LT (E112K) as adjuvant exhibited significantly (p < 0.05) enhanced levels of IL-4, IL-5, and IFN-γ as compared with T cell cultures from mice immunized with SBR alone (Fig. 5, C and D). As compared with wild-type controls, B7-2-deficient mice immunized with SBR and LT had significantly reduced levels of IL-4, IL-5, and IFN-γ, while the levels in B7-1-deficient mice were similar in magnitude (Fig. 5,C). Moreover, IL-5 and IFN-γ production from both B7-1- and B7-2-deficient mice were markedly reduced as compared with wild-type controls immunized with SBR and LT (E112K) (Fig. 5,D). While the mean levels of IL-4 production from B7-1- and B7-2-deficient mice immunized with LT (E112K) as adjuvant were also less than wild-type controls, the differences were not significant (Fig. 5 D). Similar results were observed with SBR-specific CD4+ T cells isolated from the CLN of mice immunized with LT or LT (E112K) as adjuvant (data not shown). Taken together, these results provide evidence that CD4+ T cell responses to a coadministered Ag were severely abrogated in B7-2- but not B7-1-deficient mice receiving LT as adjuvant. In contrast, the ability of LT (E112K) to augment Th1- and Th2-type cytokine production was dependent upon both B7-1 and B7-2 expression. Furthermore, B7−/− mice immunized with LT (E112K) as adjuvant lacked any discernible enhancement in SBR-specific CD4+ T cell proliferation or cytokine production as compared with mice immunized with SBR alone (data not shown).

With the aid of recombinant techniques and the development of LT mutants, it has become possible to discern the cellular mechanisms responsible for the adjuvant properties of LT. Various mutants of LT lacking ADP-ribosyltransferase activity have been shown to augment both Ab and CD4+ T cell responses to coadministered Ags (8, 9, 10, 11, 23, 24). These previous studies also provided data that the adjuvant properties of native and mutant LT differ with respect to their ability to prime mucosal and systemic CD4+ T cells. In this regard, Ryan et al. (11) demonstrated that native LT and a nonenzymatic mutant of LT induced distinct B7 up-regulation on peritoneal macrophages and suggested that these molecules may enhance different B7 molecules for their adjuvant properties. Our present study confirms and extends these observations by demonstrating not only that LT and mutant LT exhibited unique abilities to enhance B7-1 and B7-2 expression on the same APC but also that the B7 up-regulation induced by these molecules was dependent upon the type of APC. Furthermore, by using an in vitro coculture system, we have showed that the level of B7 up-regulation induced by native or mutant LT induced a functional costimulatory signal to anti-CD3-stimulated CD4+ T cells. Our in vivo experiments showing that the adjuvant activity of LT was severely abrogated in B7-2- but not B7-1-deficient mice provided support to our in vitro findings. This observation is consistent with the work of Yamamoto et al. (25) demonstrating that B7-2 up-regulation induced by native LT was largely responsible for its costimulatory properties in vitro. In contrast to native LT, we also demonstrated that the adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4+ T cell responses, while B7-1 appeared to be the predominant B7 molecule for augmenting Ab responses. Taken together, these data demonstrate not only that the up-regulation of B7 on APC by native and mutant LT differ but that the ability to up-regulate B7 molecules is a key component for their adjuvanticity.

Previous studies have shown that the B7-CD28 signaling pathway is critical for the initial activation and effector function of CD4+ T cells (13, 14, 15, 16, 17). It has also been show that B7 molecules play a critical role in both isotype switching and the formation of germinal centers (19). A study by Borriello et al. (19) assessing the humoral immune responses in B7-1- or B7-2-deficient mice suggested that B7-1 and B7-2 molecules appear to have overlapping roles when immunized with an Ag and adjuvant such as CFA or alum. However, studies using CT have shown that its ability to enhance both mucosal and systemic Ab responses to a coadministered Ag were greatly dependent upon enhancing B7-2 expression in vivo (22). Our current findings also demonstrated that both native and a mutant LT were differentially dependent upon B7-1 or B7-2 for their adjuvant properties, and that a compensatory effect was not observed in B7-1-deficient or B7-2-deficient mice. In this regard, the ability of LT to enhance humoral immune responses to a coadministered Ag was greatly reduced in B7-2- but not B7-1-deficient mice, whereas B7-1 was the main B7 molecule responsible for the ability of LT (E112K) to enhance humoral immune responses. Thus, these findings demonstrate that B7-1 and B7-2 appear to have unique costimulatory functions in the adjuvant properties of LT.

Past studies assessing the role of ADP-ribosyltransferase activity and subsequent cAMP production by LT have suggested that enzymatic activity is required for B7 up-regulation on APC (26, 27, 28, 29, 30, 31). Studies by Cong et al. (22) showed that the ability of native CT to selectively enhance B7-2 on macrophage was closely mimicked by dibutyryl cAMP, while the recombinant B subunit of CT had no effect on B7 expression. Moreover, it has been shown that cAMP can enhance B7 expression on B cells (32). While it is believed that the ADP-ribosyltransferase activity of LT does exert an effect on B7 expression, it has also been reported that a nonenzymatic mutant of LT can up-regulate B7-1 on macrophage (11). These investigators also suggested that the enzymatic activity of native LT is largely responsible for its predominant effect on B7-2 expression, while the nonenzymatic AB5 complex enhances predominantly B7-1 expression on peritoneal macrophages. Our present findings confirm and extend these observations in that native LT enhanced B7-1 and especially B7-2 expression on macrophages, whereas LT (E112K) predominantly enhanced B7-1 expression. Furthermore, LT induced mainly B7-2, while LT (E112K) induced a pronounced increase in B7-1 and to a lesser extent B7-2 expression on dendritic cells. The apparent inability of LT (E112K) to enhance either B7-1 or B7-2 expression on B cells also suggests that the ability of the nonenzymatic AB5 complex to enhance B7-1 expression may be dependent upon the type of APC. Taken together, these findings suggest that the effects of both the enzymatic activity of native LT and the nonenzymatic AB5 complex appear to have differential effects on the ability to up-regulate B7-1 and B7-2 expression on B cells, macrophages, and dendritic cells.

Our findings concerning B7-1 and B7-2 up-regulation induced by LT (E112K) are somewhat different from those reported by others (11). In this regard, it was previously shown that a nonenzymatic mutant of LT did not exhibit any discernible ability to up-regulate B7-2 expression on macrophage; however, our present findings showed that LT (E112K) was capable of enhancing both B7-1 and B7-2 expression on macrophage. A possible reason for these differences may be related to the dose of mutant LT used in the study. Assessment of B7-2 expression induced by mutant LT in our study demonstrated no significant effect on B7-2 expression until concentrations of 1 μg/ml or higher were used. Thus, our findings provide evidence that there is a dose-dependent effect on B7-2 expression induced by mutant LT. This may account for our observations that LT (E112K) can enhance B7-2 expression on macrophage. Moreover, while the present study demonstrated that the ability of native and mutant LT to enhance B7 expression on APC was different and likely related to enzymatic activity, our findings concerning LT (E112K) demonstrate that the ability to up-regulate B7 expression is not solely dependent upon ADP-ribosyltransferase activity.

The present study provides evidence that the adjuvanticity of both the native LT and the mutant LT (E112K) is in part mediated by their ability to enhance B7-1 and/or B7-2 expression. While both native and mutant LT enhanced B7 expression on APCs, their dependence upon the B7 costimulatory pathway for their adjuvant properties appears to differ. The ability of LT (E112K) to augment CD4+ T cell responses was strongly dependent on the ability to enhance both B7-1 and B7-2 expression. Indeed, CD4+ T cell responses in B7-1- or B7-2-deficient mice immunized with LT (E112K) as adjuvant did not exhibit any significant enhancement in proliferation or cytokine production, as compared with mice immunized with Ag alone. In contrast, while our data show that the adjuvant properties of LT were, in part, dependent upon the ability to enhance B7-2 expression, LT was still able to significantly enhance CD4+ T cell responses and cytokine production in B7-2-deficient mice as compared with mice immunized with Ag alone. Preliminary studies using LT as adjuvant demonstrated that LT does retain a partial ability to enhance CD4+ T cell responses to a coadministered Ag in B7-deficient mice (our unpublished observations), which is likely dependent upon the ability of native LT to enhance cAMP levels. Previous studies have shown that cAMP can affect a variety of costimulatory pathways that may partially overcome the need for B7 costimulation of CD4+ T cells (32, 33).

The ability of B7-1 and B7-2 to deliver unique costimulatory signals and directly influence Th1 and Th2 development remains an unresolved issue. Initial studies suggested that B7-1 and B7-2 preferentially activated Th1 and Th2 cells, respectively (13, 15, 16, 34, 35). However, it was subsequently reported that B7-1 and B7-2 deliver similar costimulatory signals to T cells and thus do not appear to selectively promote Th1 or Th2 phenotypes (14, 36). Our present findings suggest that native LT enhanced B7-2 expression for the induction of both Th1- and Th2-type immune responses. Analysis of the SBR-specific CD4+ T cell cytokine profiles from B7-2-deficient mice immunized with native LT as adjuvant revealed reduced levels of both Th1-associated (IFN-γ) and Th2-associated (IL-4, IL-5) cytokines. Moreover, due to the involvement of IL-4 and IFN-γ influencing IgG1 and IgG2a Ab responses, respectively (37, 38), our data concerning anti-SBR IgG1 and IgG2a Ab responses in B7-2-deficient mice further support our evidence that the ability of native LT to up-regulate B7-2 results in enhancing both Th1- and Th2-mediated immune responses. These findings support those of a previous study by Yamamoto et al. (25), who demonstrated that native LT promotes both Th1 and Th2 cells by enhancing B7-2 and not B7-1 expression on B cells and macrophage. Interestingly, we also provide data that LT (E112K) enhanced SBR-specific IgG1 and IgG2a Abs predominantly via the ability to increase B7-1 expression in vivo, while both B7-1 and B7-2 expression were required for optimal Th1 and Th2 cytokine production from CD4+ T cells. Thus, while the ability of LT and LT (E112K) to up-regulate B7 expression is involved in their ability to enhance Ag-specific Ab and CD4+ T cell responses to a coadministered Ag, there does not appear to be an association between the ability of LT or LT (E112K) to selectively influence Th1 or Th2 development via B7-1 or B7-2, respectively.

In summary, we have demonstrated that the adjuvant properties of LT and LT (E112K) are differentially influenced by their ability to up-regulate B7-1 and B7-2 expression. These studies provide insight into the cellular mechanisms responsible for the ability of LT to act as a mucosal and systemic adjuvant.

1

These studies were supported by U.S. Public Health Service Grants DE14215, DE08182, and DE09081 (to S.M.M.), and HL 07553 (to M.M.).

3

Abbreviations used in this paper: LT, heat-labile enterotoxin of Escherichia coli; CT, cholera toxin; i.n., intranasal(ly); SBR, saliva binding region; CLN, cervical lymph node; MFI, mean fluorescence intensity; NALT, nasal-associated lymphoid tissue.

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